An albumin-binding domain as a scaffold for bispecific affinity - DiVA

115 Pages·2012·7.25 MB·English

An albumin-binding
domain as a scaffold for
bispecific affinity proteins
Johan nilveBranT
Doctoral Thesis in Biotechnology
Stockholm, Sweden 2012
An albumin-binding domain as a scaffold An albumin-binding domain as a scaffold
for bispecific affinity proteins for bispecific affinity proteins

JOHAN NILVEBRANT JOHAN NILVEBRANT

Royal Institute of Technology Royal Institute of Technology
School of Biotechnology School of Biotechnology
Stockholm 2012 Stockholm 2012
© Johan Nilvebrant © Johan Nilvebrant
Stockholm 2012 Stockholm 2012

Royal Institute of Technology Royal Institute of Technology
School of Biotechnology School of Biotechnology
AlbaNova University Center AlbaNova University Center
SE-106 91 Stockholm SE-106 91 Stockholm
Sweden Sweden

Printed by E-Print Printed by E-Print
Oxtorgsgatan 9 Oxtorgsgatan 9
SE-111 57 Stockholm SE-111 57 Stockholm
Sweden Sweden

ISBN 978-91-7501-569-9 ISBN 978-91-7501-569-9
TRITA-BIO Report 2012:21 TRITA-BIO Report 2012:21
ISSN 1654-2312 ISSN 1654-2312
iii iii

Johan Nilvebrant (2012): An albumin-binding domain as a scaffold for bispecific affinity Johan Nilvebrant (2012): An albumin-binding domain as a scaffold for bispecific affinity
proteins. proteins.
Division of Proteomics, School of Biotechnology, Royal Institute of Technology (KTH), Stockholm, Division of Proteomics, School of Biotechnology, Royal Institute of Technology (KTH), Stockholm,
Sweden. Sweden.
Abstract Abstract
Protein engineering and in vitro selection systems are powerful methods to generate binding proteins. Protein engineering and in vitro selection systems are powerful methods to generate binding proteins.
In nature, antibodies are the primary affinity proteins and their usefulness has led to a widespread use In nature, antibodies are the primary affinity proteins and their usefulness has led to a widespread use
both in basic and applied research. By means of combinatorial protein engineering and protein library both in basic and applied research. By means of combinatorial protein engineering and protein library
technology, smaller antibody fragments or alternative non-immunoglobulin protein scaffolds can be technology, smaller antibody fragments or alternative non-immunoglobulin protein scaffolds can be
engineered for various functions based on molecular recognition. In this thesis, a 46 amino acid small engineered for various functions based on molecular recognition. In this thesis, a 46 amino acid small
albumin-binding domain derived from streptococcal protein G was evaluated as a scaffold for the albumin-binding domain derived from streptococcal protein G was evaluated as a scaffold for the
generation of affinity proteins. Using protein engineering, the albumin binding has been generation of affinity proteins. Using protein engineering, the albumin binding has been
complemented with a new binding interface localized to the opposite surface of this three-helical complemented with a new binding interface localized to the opposite surface of this three-helical
bundle domain. By using in vitro selection from a combinatorial library, bispecific protein domains bundle domain. By using in vitro selection from a combinatorial library, bispecific protein domains
with ability to recognize several different target proteins were generated. In paper I, a bispecific with ability to recognize several different target proteins were generated. In paper I, a bispecific
albumin-binding domain was selected by phage display and utilized as a purification tag for highly albumin-binding domain was selected by phage display and utilized as a purification tag for highly
efficient affinity purification of fusion proteins. The results in paper II show how protein engineering, efficient affinity purification of fusion proteins. The results in paper II show how protein engineering,
in vitro display and multi-parameter fluorescence-activated cell sorting can be used to accomplish the in vitro display and multi-parameter fluorescence-activated cell sorting can be used to accomplish the
challenging task of incorporating two high affinity binding-sites, for albumin and tumor necrosis challenging task of incorporating two high affinity binding-sites, for albumin and tumor necrosis
factor-alpha, into this new bispecific protein scaffold. Moreover, the native ability of this domain to factor-alpha, into this new bispecific protein scaffold. Moreover, the native ability of this domain to
bind serum albumin provides a useful characteristic that can be used to extend the plasma half-lives bind serum albumin provides a useful characteristic that can be used to extend the plasma half-lives
of proteins fused to it or potentially of the domain itself. When combined with a second targeting of proteins fused to it or potentially of the domain itself. When combined with a second targeting
ability, a new molecular format with potential use in therapeutic applications is provided. The ability, a new molecular format with potential use in therapeutic applications is provided. The
engineered binding proteins generated against the epidermal growth factor receptors 2 and 3 in papers engineered binding proteins generated against the epidermal growth factor receptors 2 and 3 in papers
III and IV are aimed in this direction. Over-expression of these receptors is associated with the III and IV are aimed in this direction. Over-expression of these receptors is associated with the
development and progression of various cancers, and both are well-validated targets for therapy. development and progression of various cancers, and both are well-validated targets for therapy.
Small bispecific binding proteins based on the albumin-binding domain could potentially contribute Small bispecific binding proteins based on the albumin-binding domain could potentially contribute
to this field. The new alternative protein scaffold described in this thesis is one of the smallest to this field. The new alternative protein scaffold described in this thesis is one of the smallest
structured affinity proteins reported. The bispecific nature, with an inherent ability of the same structured affinity proteins reported. The bispecific nature, with an inherent ability of the same
domain to bind to serum albumin, is unique for this scaffold. These non-immunoglobulin binding domain to bind to serum albumin, is unique for this scaffold. These non-immunoglobulin binding
proteins may provide several advantages as compared to antibodies in several applications, proteins may provide several advantages as compared to antibodies in several applications,
particularly when a small size and an extended half-life are of key importance. particularly when a small size and an extended half-life are of key importance.

Keywords: albumin-binding domain, bispecific, albumin, affinity protein, phage display, Keywords: albumin-binding domain, bispecific, albumin, affinity protein, phage display,
staphylococcal display, orthogonal affinity purification, TNF-α, ErbB2, ErbB3 staphylococcal display, orthogonal affinity purification, TNF-α, ErbB2, ErbB3

© Johan Nilvebrant 2012 © Johan Nilvebrant 2012
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Papers in this thesis Papers in this thesis

This thesis is based on the following papers, which are referred to in the text by their This thesis is based on the following papers, which are referred to in the text by their
Roman numerals (I-IV). They are included in the appendix. Roman numerals (I-IV). They are included in the appendix.

I Alm, T., Yderland, L., Nilvebrant, J., Halldin, A., Hober, S. (2010). A small I Alm, T., Yderland, L., Nilvebrant, J., Halldin, A., Hober, S. (2010). A small
bispecific protein selected for orthogonal affinity purification, Biotechnol. J., 5: bispecific protein selected for orthogonal affinity purification, Biotechnol. J., 5:
605-617 605-617

II Nilvebrant, J., Alm, T., Hober, S., Löfblom, J. (2011). Engineering bispecificity II Nilvebrant, J., Alm, T., Hober, S., Löfblom, J. (2011). Engineering bispecificity
into a single albumin-binding domain, PLoS ONE, 6(10): e25791 into a single albumin-binding domain, PLoS ONE, 6(10): e25791

III Nilvebrant, J., Åstrand, M., Georgieva, M., Björnmalm, M., Löfblom, J., Hober, III Nilvebrant, J., Åstrand, M., Georgieva, M., Björnmalm, M., Löfblom, J., Hober,
S. Engineering of bispecific affinity proteins with nanomolar affinity for both S. Engineering of bispecific affinity proteins with nanomolar affinity for both
ErbB2 and albumin, manuscript ErbB2 and albumin, manuscript

IV Nilvebrant*, J., Åstrand*, M., Löfblom, J., Hober, S. Development and IV Nilvebrant*, J., Åstrand*, M., Löfblom, J., Hober, S. Development and
characterization of small bispecific three-helical ErbB3/albumin-binding domains characterization of small bispecific three-helical ErbB3/albumin-binding domains
aimed at therapeutic applications, manuscript aimed at therapeutic applications, manuscript

* Authors contributed equally * Authors contributed equally

All papers and figures derived from them have been reproduced with permission from All papers and figures derived from them have been reproduced with permission from
copyright holders. copyright holders.
v v
Papers not included in the thesis Papers not included in the thesis

Boström*, T., Nilvebrant* J., Hober, S. (2012). Purification systems based on bacterial Boström*, T., Nilvebrant* J., Hober, S. (2012). Purification systems based on bacterial
surface proteins. Protein Purification, R. Ahmad (Ed.), ISBN: 978-953-307-831-1, InTech surface proteins. Protein Purification, R. Ahmad (Ed.), ISBN: 978-953-307-831-1, InTech

Nilvebrant, J., Alm, T., Hober, S. (2012). Orthogonal protein purification facilitated by a Nilvebrant, J., Alm, T., Hober, S. (2012). Orthogonal protein purification facilitated by a
small bispecific affinity tag. J. Vis. Exp. (59), e3370 small bispecific affinity tag. J. Vis. Exp. (59), e3370

Nilvebrant, J., Dunlop, C., Wurch, T., Falkowska, E. Helguera, G., Piccione, E., Reichert, Nilvebrant, J., Dunlop, C., Wurch, T., Falkowska, E. Helguera, G., Piccione, E., Reichert,
J. (2012). Meeting report of the 2011 IBC’s 22nd Annual International Conference on J. (2012). Meeting report of the 2011 IBC’s 22nd Annual International Conference on
Antibody Engineering and Antibody Therapeutics, mAbs, 4(2): 153-181 Antibody Engineering and Antibody Therapeutics, mAbs, 4(2): 153-181

Nilvebrant, J., Kuku, G., Björkelund, H., Nestor, M. (2012). Selection and in vitro Nilvebrant, J., Kuku, G., Björkelund, H., Nestor, M. (2012). Selection and in vitro
characterization of human CD44v6-binding antibody-fragments, Biotech. Appl. Biochem., characterization of human CD44v6-binding antibody-fragments, Biotech. Appl. Biochem.,
59(5): 367-380 59(5): 367-380

* Authors contributed equally * Authors contributed equally
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“A classic is something that everyone wants to have read and nobody wants to read” “A classic is something that everyone wants to have read and nobody wants to read”

– Mark Twain – Mark Twain
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CONTENTS CONTENTS
INTRODUCTION....................................................................................................1 INTRODUCTION....................................................................................................1

1. PROTEIN ENGINEERING.................................................................................2 1. PROTEIN ENGINEERING.................................................................................2
1.1 Rational protein engineering.....................................................................................5 1.1 Rational protein engineering.....................................................................................5
1.2 Combinatorial protein engineering..........................................................................9 1.2 Combinatorial protein engineering..........................................................................9

2. IN VITRO SELECTION SYSTEMS..................................................................13 2. IN VITRO SELECTION SYSTEMS..................................................................13
2.1 Cell-dependent systems............................................................................................17 2.1 Cell-dependent systems............................................................................................17
2.1.1 Phage display................................................................................18 2.1.1 Phage display................................................................................18
2.1.2 Cell-display...................................................................................20 2.1.2 Cell-display...................................................................................20
2.1.2.1 Yeast display..............................................................................23 2.1.2.1 Yeast display..............................................................................23
2.1.2.2 E. coli display.............................................................................24 2.1.2.2 E. coli display.............................................................................24
2.1.2.3 Staphylococcal display...............................................................25 2.1.2.3 Staphylococcal display...............................................................25
2.2 Cell-independent systems........................................................................................26 2.2 Cell-independent systems........................................................................................26
2.3 Non-display systems.................................................................................................27 2.3 Non-display systems.................................................................................................27

3. AFFINITY PROTEINS AND PROTEIN SCAFFOLDS FOR MOLECULAR 3. AFFINITY PROTEINS AND PROTEIN SCAFFOLDS FOR MOLECULAR
RECOGNITION.....................................................................................................29 RECOGNITION.....................................................................................................29
3.1 Antibodies and antibody derivatives......................................................................30 3.1 Antibodies and antibody derivatives......................................................................30
3.2 Alternative, non-immunoglobulin, scaffold proteins............................................34 3.2 Alternative, non-immunoglobulin, scaffold proteins............................................34
3.2.1 Anticalins......................................................................................40 3.2.1 Anticalins......................................................................................40
3.2.2 Designed ankyrin repeat proteins..................................................40 3.2.2 Designed ankyrin repeat proteins..................................................40
3.2.3 Adnectins.......................................................................................41 3.2.3 Adnectins.......................................................................................41
3.2.4 Affibody molecules and other affinity proteins derived from 3.2.4 Affibody molecules and other affinity proteins derived from
the Z-domain..........................................................................................42 the Z-domain..........................................................................................42
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4. THE ALBUMIN-BINDING DOMAIN (ABD), HOMOLOGS AND 4. THE ALBUMIN-BINDING DOMAIN (ABD), HOMOLOGS AND
ENGINEERED VARIANTS..................................................................................46 ENGINEERED VARIANTS..................................................................................46
4.1 In vivo half-life extension and its relation to ABD................................................52 4.1 In vivo half-life extension and its relation to ABD................................................52

5. PRESENT INVESTIGATION...........................................................................58 5. PRESENT INVESTIGATION...........................................................................58
5.1 Aim............................................................................................................................58 5.1 Aim............................................................................................................................58
5.2 ABD as a scaffold for protein engineering, design of a library for selection of 5.2 ABD as a scaffold for protein engineering, design of a library for selection of
bispecific domains and proof of principle....................................................................58 bispecific domains and proof of principle....................................................................58
5.3 Engineering of high affinity bispecific binding proteins based on ABD.............65 5.3 Engineering of high affinity bispecific binding proteins based on ABD.............65
5.4 ABD-based targeting of cancer-related members of the human epidermal 5.4 ABD-based targeting of cancer-related members of the human epidermal
growth factor receptor family.......................................................................................70 growth factor receptor family.......................................................................................70

6. CONCLUSIONS AND FUTURE OUTLOOK..................................................79 6. CONCLUSIONS AND FUTURE OUTLOOK..................................................79
Acknowledgements.........................................................................................................84 Acknowledgements.........................................................................................................84

7. REFERENCES...................................................................................................87 7. REFERENCES...................................................................................................87
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Common abbreviations Common abbreviations

ABD Albumin-binding domain derived from streptococcal protein G (G148-ABD3) ABD Albumin-binding domain derived from streptococcal protein G (G148-ABD3)

ALB8-GA Albumin-binding domain derived from protein albumin binding of F. magna ALB8-GA Albumin-binding domain derived from protein albumin binding of F. magna

CDR Complementarity determining region CDR Complementarity determining region

ErbB2 Epidermal growth factor receptor 2 (HER2) ErbB2 Epidermal growth factor receptor 2 (HER2)

ErbB3 Epidermal growth factor receptor 3 (HER3) ErbB3 Epidermal growth factor receptor 3 (HER3)

Fab Fragment antigen binding (of an antibody molecule) Fab Fragment antigen binding (of an antibody molecule)

FACS Fluorescence-activated cell sorting FACS Fluorescence-activated cell sorting

Fc Fragment crystallizable (of an antibody molecule) Fc Fragment crystallizable (of an antibody molecule)

FcRn Neonatal Fc-receptor (Brambell receptor) FcRn Neonatal Fc-receptor (Brambell receptor)

HSA Human serum albumin HSA Human serum albumin

IgG Immunoglobulin G IgG Immunoglobulin G

K Dissociation equilibrium constant K Dissociation equilibrium constant
D D

mAb Monoclonal antibody mAb Monoclonal antibody

scFv Single-chain fragment variable scFv Single-chain fragment variable

SPA Staphylococcal protein A SPA Staphylococcal protein A

SPG Streptococcal protein G SPG Streptococcal protein G

SPR Surface plasmon resonance SPR Surface plasmon resonance

TNF-α Tumor necrosis factor-alpha TNF-α Tumor necrosis factor-alpha